Neuronal nicotinic acetylcholine receptors (nAChRs) are transmitted gated ion channels that belong to a gene super family of homologous receptors including γ-aminobutyric acid (GABA), glycine, and serotonin receptors. The receptor is composed of five subunits with each having four transmembrane domains (M1-M4) and a large intracellular loop (C2). The genes for the subunits that have been cloned so far are divided into two subfamilies of nine α (α2-α10) and three β (β2-β4) subunits, and are expressed in the nervous system, cochlea, and a number of non-neuronal tissues.
Neuronal nAChRs have a role in the mediation of tolerance and addiction to nicotine in chronic tobacco users and the symptoms of withdrawal experienced upon cessation of use. The complex activities of nicotine in the nervous system are due to its ability to mimic the activity of acetylcholine (ACh) on these receptors. At the molecular level, chronic nicotine exposure differentially affects the number (up-regulation), subunit composition, stoichiometry, and functional status (desensitization and inactivation) of some nAChR subtypes, leaving others substantially unaffected. Nicotine, the addictive component of tobacco, has a predominant effect in the brain mainly on the α4β2 nAChR, the most abundant subtype.
Desensitization and up-regulation of nAChRs is thought to involve phosphorylation mediated by various kinases. For instance, protein kinase A (PKA) and protein kinase C (PKC) are examples of kinases that have been studied in the context of nAChR desensitization and up-regulation. Specifically, studies suggest that in the continuous presence of nicotine, receptors would be driven into inactive/desensitized conformations, a process likely influenced by the level of phosphorylation. There is evidence that activators of PKA and PKC increase nAChR binding sites and synergistically enhance nicotine-induced receptor up-regulation. These findings are validated by in vitro and in vivo studies, which have demonstrated that α4 nAChR subunits are phosphorylated and, more specifically, that they are targets of PKA phosphorylation. Studies using phosphopeptide mapping have provided evidence that residues S365, S472, and S491 of the rat α4 subunit, corresponding to positions S364, S471 and S490 on the current α4 NCBI reference sequence, are substrates for PKA phosphorylation. In addition, a recent study identified two major substrate sites for PKA phosphorylation on the human α4 subunit, namely S467 and S362, which are homologues to rat α4 positions S471 and S364 used in the current study.
Recent work from the inventors has shown that two-point mutations of a PKC phosphorylation residue, S336A and S336D, exhibit constitutive up-regulation when expressed in oocytes. In addition, we found that both of these PKC mutations changed the ACh affinity but exhibited no change in nicotine affinity, suggesting that the properties of agonist binding for α4β2 channel activation have very distinct dynamic and/or structural requirements for ACh and nicotine.